Abstract Epstein-Barr virus (EBV), the first identified human tumor virus, transforms resting B-cells into rapidly growing lymphoblasts. EBV-driven lymphomas cause significant disease in patients with primary or acquired immunodeficiency, including post-transplantation, with HIV co-infection or with age-related immune-senescence. EBV transforms human B-cells through a program that involves at least three early stages. First, EBV causes major B-cell anabolic remodeling, prior to cell cycle entry. Second, EBV then drives rapid Burkitt-lymphoma (BL) like B-cell growth, triggering cell division every 8-12 hours. Finally, EBV LMP1 and 2A oncoprotein expression cause a transition to lymphoblastoid cell line (LCL)-like growth, where cells divide daily and complete the immortalization program. To achieve this remarkable transition, EBV subverts key host metabolic networks to provide energy, biosynthetic building blocks and protection from reactive oxygen species. Yet, comprehensive approaches have not been used to identify key EBV-targeted mitochondrial metabolic pathways that underlie EBV-mediated B-cell growth transformation. We therefore used multiplexed mass spectrometry to create a temporal proteomic map of EBV-mediated primary human B-cell transformation, and used genome-wide CRISPR screens to identify EBV-induced B-cell dependency factors. These approaches identified a central role for EBV-induced mitochondrial one-carbon (1C) metabolism, an embryonic program that enables rapid cell growth but which is shut off in many adult tissues. Our preliminary data indicates that EBV oncoproteins induce 1C metabolism, including in a mouse model of lymphoproliferative disease. Our central hypothesis is that EBV-induced mitochondrial one-carbon metabolism has critical but distinct roles in each stage of EBV- mediated B-cell growth transformation. Our Specific Aims are therefore to: 1) Identify the role of EBV-induced 1C metabolism in primary B-cell remodeling; (2) Identify the role of one-carbon metabolism in support of rapid Burkitt-like growth; (3) Identify mitochondrial 1C roles in LCL-like cell redox defense. Collectively, these studies are expected to identify how EBV subverts a key embryonic mitochondrial pathway to enable potent growth transformation. 1C metabolism has not been studied in EBV pathogenesis, but is closely related to host enzyme targets blocked by antifolate lymphoma therapies. Our studies may therefore support strategies to develop rational therapeutic regimens to halt EBV-associated malignancies.